Investigation of chain-length selection by the tenellin iterative highly-reducing polyketide synthase

The programming of widely distributed iterative fungal hr-PKS is mysterious, yet it is central for generating polyketide natural product diversity by controlling the chain length, β-processing level and methylation patterns of fungal polyketides. For the iterative hr-PKS TENS, responsible for producing the pentaketide–tyrosine hybrid pretenellin A 1, the chain length programming is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of swap experiments with corresponding sequences from the related hr-PKS DMBS and MILS that produce similar hexa- and heptaketides (2, 3). The investigations of chimeric TENS variants expressed in vivo in the host Aspergillus oryzae NSAR1 revealed the substrate binding helix as a promising target for further investigations, evidenced by observed increase of the chain length during swap experiments. Building on these findings, rational engineering of TENS was applied based on structural analysis and sequence alignment. A minimal set of four simultaneous amino acid mutations achieved the re-programming of TENS by producing hexaketides in minor amounts. To refine our understanding and minimize the number of mutations impacting polyketide chain length, we conducted an alanine scan, pinpointing crucial amino acid positions. Our findings give indications on the intrinsic programming of hr-PKS domains by minimal changes in the amino acid sequence as one influence factor for programming.


Cloning Procedure, Vectors and Oligonucleotides
The strategy for vector construction (Figure S1) was based on yeast homologous recombination.The enzymes (EcoRI, FseI) used for vector digestion within the tenS gene were purchased from New England Biolabs (Beverly, MA, USA) and used according to the manufacturer's instructions with appropriate supplied buffers.
The proofreading Q5 ® 2x Master Mix (New England Biolabs) was used to obtain DNA fragments needed for further cloning procedure (F1-F3) with the pEYA-tenS as a template.The information provided by the manufacturer served as a template.Mutations/swap sequences were introduced by synthetic fragments (Twist Bioscience) to rebuild pEYA-tenS*.The transformation of S. cerevisiae was done using the LiOAc/SS carrier DNA/PEG protocol developed by Gietz and Woods. 1,2Therefore, fresh yeast cells were incubated with the transformation-mix (50 µl 2 mg/ml ssDNA, 240 µl 50 % PEG 3350, 36 µl 1 M LiAc, up to 5 µg DNA (cut plasmid, fragments, equimolar)) for 42 °C for 50 min.Cells were pelleted at 11000 x g for 30 s and resuspended with 500 µl water.250 µl of the cell mixture was spread on selective SM-URA plates and incubated for 3-5 days at 30 °C.The vector DNA was then purified from yeast, transformed into E. coli Top 10.DNA samples were sequenced by Eurofins Genomics (Mix2Seq OVERNIGHT, Ebersberg).
Gateway LR Clonase II Enzyme mix kit (Invitrogen) 3 was used to transfer genes from the entry vector pEYA-tenS* to the destination vector pTY-GS-argB-tenC (Figure S1).The manufacturer's instructions were followed.For E.coli Top10 transformation, the vector mixture (10 µl) was added to 50 µl competent cells.
Information about the used vectors in this work are summarised in Table S1.

Strains and Cultivation
Information about the used strains in this work are summarised in Table S3.Antibiotics (carbenicillin, kanamycin) were prepared in 1000x concentrated stock solution in distilled water in a concentration of 50 µM.Stocks were sterilized through 0.45 µm syringe filter and stored at -20 °C.Antibiotics were diluted for a final concentration of 50 µg/ml by adding them to the final media.

Growth and Maintenance
E. coli cells were grown on LB-agar or in liquid LB-medium with corresponding antibiotics.The cells were cultivated at 37 °C for approx.12 h.If grown in liquid media, the culture was shaking at 200 rpm.
S. cerevisiae cells were grown on solid YPAD agar at 30 °C for 3 -5 days.One single colony was used for inoculation for 10 ml liquid YPAD medium.The culture was grown overnight at 30 °C and 200 rpm.After transformation of cells with vectors containing ura3 selection marker the cultivation took place with selective SM-URA-Agar and at 30°C for 3-5 days.
A. oryzae strain NSAR1 was grown on DPY-agar plates for 3-7 days at 28 °C. A. oryzae strain NSAR1 transformants were grown in 100 ml CMP liquid medium in 500 ml baffled shake flasks for 6 days at 28 °C and 110 rpm.

Transformations
Competent E.coli strains were thawed on ice after -80 °C storage.60 -100 ng purified plasmid was added to 50 µl E.coli cells and placed on ice for 30 min, followed by a heat shock at 42 °C for 30 s and cooling on ice for 2 min.250 µl SOC-medium was added to the cells and the mixture was incubated at 37°C and 300 rpm for 1 h.The transformed cells were spread out on LB-agar plates containing appropriate antibiotics and incubated at 37 °C overnight.
A. oryzae NSAR1 was grown on DPY-plates at 28 °C for 5-7 days.The mycelium was used to inoculate 50 ml GN-medium in 250 ml shake flask.The culture was incubated at 28 °C and 110 rpm for approx.18 h.The biomass was separated from the media by filtration with a miracloth filter.Mycelia was incubated with VinoTaste® Pro (Novozymes) solution (10 mg/ml enzyme) while shaking at room temperature and 2 rpm for 3-5 hours.The protoplasts were obtained by filtration with a miracloth filter and centrifuged at 3000 x g for 5 min.The resulting pellet was re-suspended with solution 1 (100 µl per transformation, 0.8 M sodium chloride, 10 mM calcium chloride, 50 mM Tris-HCl, pH 7.5).Vector DNA was added to 100 µl protoplast suspension and incubated on ice for 2 min.Then 1 ml of solution 2 (60 % (w/v) PEG 3350, 0.8 M sodium chloride, 10 mM calcium chloride, 50 mM Tris-HCl, pH 7.5) was added to the mixture and incubated at room temperature for 20 min.After incubation 5 ml of appropriate selective softagar was added to the mixture and overlaid over prepared plates with corresponding agar.Plates were incubated at 28 °C for 4-6 days.When mycelia was visible, the transformants undergo two further selection rounds to avoid false-positive transformants.The colonies are picked from the agar, placed on fresh selection agar plates, and grown for 3-5 days.For the preparation of liquid cultures the transformants were grown on DPY agar plates for 5 days.The spores were used to inoculate 100 ml CMP liquid medium.

Analytical LCMS
Analytical LCMS was run to analyse the extracts from fungal cultures.The Waters LCMS system containing a Waters 2767 autosampler, Waters 2545 pump, a Phenomenex Kinetex column (2.6 μm, C18, 100 Å, 4.6 x 100 mm), a Phenomenex Security Guard precolumn (Luna, C5, 300 Å) was used with a flow rate of 1 ml/min.The equipped detectors were a diode array detector (Waters 2998) in the range 210 to 600 nm and an ELSD detector (Waters 2424) together with a mass spectrometer, Waters SQD-2 mass detector (ES + and ES -, 150 and 1000 m/z).For elution, a solvent gradient was run for 15 min starting at 10 % acetonitrile/ 90 % HPLC grade water (0.05 % formic acid) and ramping to 90 % acetonitrile.

Figure S4 .
Figure S4.Overlay of TENS KR model (green-yellow), AmphB KR (cyan) and Tylosin KR1 (magenta) showing location of cofactor (grey), Amph B substrate (cyan) and TENS substrate (magenta).The substrate-binding helix (sbh) of the TENS KR is indicated in yellow and overlays well with the 'lid-helix' of the Amph B structure, but the lid-helix of the Tylosin structure approaches the bound substrates more closely.Distances calculated between amide nitrogen of N-terminal residues of the helices and the amide nitrogen of the active site tyrosine.

Table S1
Summary of used vectors.

Table S3
Summary of used strains.TableS4), buffers and solutions used in this work were prepared with Millipore water (GenPure Pro UV/UF milipore device, Thermo Fisher Scientific) and sterilised by autoclaving 15 min at 121 °C (Autoclave 2100 Classic, Prestige Medical) or sterilised by disposable syringe filters (pore size 0.2 -0.4 µm, Carl Roth).The pH was adjusted with 2 M HCl or 2 M NaOH by using a FiveEasy Standard pH Meter Line (Mettler Toledo).

Table S4
Media used in this work.

Table S5
Structural comparison and resulting RMSD of LovB with threaded and AlphaFold model.